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Fig. 2 | Microbiome

Fig. 2

From: Simple discovery of bacterial biocatalysts from environmental samples through functional metaproteomics

Fig. 2

ML-005 is a novel esterase. a ML-005 is a distant relative (35% identity) of the uncharacterized putative hydrolase YdeN of B. subtilis. b Heterologous protein expression of ML-005 and lipase A LipA from B. subtilis (positive control) in E. coli from a plasmid was induced with 1 mM IPTG. E. coli carrying the empty vector served as negative control. Cells were disrupted by sonication and crude extracts were subjected to SDS PAGE, the protein content visualized by coomassie staining. c Lipid hydrolyzing activity was detected through in-gel zymography in the same crude cell lysates. The in-gel activity assay shows substrate conversion for positive control LipA from B. subtilis (23 kDa) and ML-005 (24.5 kDa) while a negative control of an extract of E. coli carrying the empty vector shows no activity. d Crude extract of E. coli expressing ML-005 hydrolyzes para-nitrophenyl-butyrate. Crude extract of E. coli expressing LipA from B. subtilis served as positive control, crude extract of E. coli containing the empty vector as negative control. Representative results are shown, results of all biological replicates can be found in Additional file 1: Figure S4. d Substrate specificity of purified ML-005 indicates a preference towards short-chain (C4) and medium-chain length (C8) para-nitrophenyl esters typical for esterases, no activity towards long-chain (C16) esters could be detected (n.d.). Specific activity of ML-005 towards para-nitrophenyl butyrate was 14.1 U mg−1

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